Peroxisomes are ubiquitous eukaryotic organelles involved in a variety of important metabolic pathways. In humans, peroxisomes are essential for the metabolism of certain lipids, and defects in their biogenesis are responsible for a family of lethal genetic disorders, collectively termed Zellweger syndrome. The primary long-term goal of this program is to understand, at the molecular level, the mechanisms controlling peroxisomal biogenesis. A combined molecular genetic and biochemical attack has been initiated using the years Pichia pastoris as the model system. In recent years, more than a dozen different PEX genes and their products have been described. However, convincing evidence for a specific role in biogenesis has been provided for only a few of these. A major goal of this proposal is to establish the specific function of two P. pastoris PEX proteins, Pex2p and Pex8p, in peroxisome biogenesis. Pex8p is a protein located on the inner membrane of the peroxisome that may function to release newly imported matrix proteins from their receptors. Evidence for this model will be obtained by identifying and dissecting domains on Pex8p responsible for import of specific subsets of peroxisomal proteins and for interaction with specific import receptors. Pex2p belongs to a family of related peroxisomal integral membrane proteins and is the P. pastoris ortholog of a Zellweger gene. Members of this family are hypothesized to form the core of the peroxisomal protein translocation apparatus. Pulse-chase studies will be conducted on temperature-sensitive pex2 mutants to obtain evidence that Pex2p function is required for protein import. The organization of the putative import apparatus will be elucidated through the identification of Pex2p interacting proteins. A combination of approaches involving the two-hybrid system, suppressor mutant analysis and purification of Pex2p-containing complexes will be employed. Finally, a highly efficient selection scheme will be utilized to identify P. pastoris mutants defective in novel PEX genes. The PEX genes will be cloned, and the primary sequences of their products will be used to search the databases for their human PEX cDNA orthologs. Human PEX cDNAs that are also Zellweger genes will be identified by their ability to restore normal peroxisomes to specific Zellweger cell lines. Thus, this yeast system provides an excellent model for investigating peroxisome biogenesis and understanding the molecular etiology of this disease.